Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Kevin G. Chen; Rebecca S. Hamilton; Pamela G. Robey; Barbara S. Mallon

doi:10.3791/51519

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Method Article

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

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DOI:

10.3791/51519

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July 24th, 2014

Kevin G. Chen¹ , Rebecca S. Hamilton¹ , Pamela G. Robey² , Barbara S. Mallon¹

¹NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, ²Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health

Summary

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Abstract

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Introduction

The capacity of hPSCs to differentiate toward multilineage adult tissues has opened new avenues to treating patients who suffer from severe diseases that involve cardiovascular, hepatic, pancreatic, and neurological systems^1-4. Various cell types derived from hPSCs would also provide robust cellular platforms for disease modeling, genetic engineering, drug screening, and toxicological testing^1,4. The key issue that ensures their future clinical and pharmacological applications is the generation of large numbers of clinical-grade hPSCs through in vitro cell culture. However, current culture systems are either insufficient or inherently va....